Proteolytic enzymes are proteases or peptidases, i.e. enzymes that are able to split or lyse peptides or proteins. The word “peptide” generally denotes a shorter amino acid sequence, whereas the word “protein” generally denotes larger molecules consisting of an amino acid sequence. Both terms are used synonymously in the following.
Peptidases and proteases play a decisive role in protein activation, cell regulation and signal transmission. Their detection at maximum possible sensitivity is therefore of enormous importance for understanding the functioning of a cell and its communication, and for monitoring the course of diseases.
Furthermore, precise knowledge of peptidase or protease concentration permits the targeted development of new therapeutic agents for inhibition, e.g. of HIV protease. HIV protease breaks down the HIV protein, which is at first very long after multiplication in a cell, into functioning individual proteins, which only then permit the further activity and growth of the HIV virus. There are two possible approaches. Firstly, if the HIV protease is blocked, the HIV virus cannot replicate and its propagation is halted. Secondly, infection with the HIV virus can take place via detection of the HIV protease. Specific detection of HIV protease can therefore serve as an AIDS test.
Moreover, protease tests are attracting increasing attention in medical research, because more and more diseases, including cancers, are being connected with enzymes. Tumor-associated proteases are increasingly the object of oncological-biochemical research. These proteases bring about the breakdown of proteins of the tumor stroma and of the basal membrane and thus permit tumor cell invasion. Therefore investigations of protease systems that are overexpressed in tumor cells are among the new prognosis factors in tumor diagnostics.
Another important aspect is the use of protease tests in quality control of biochemical products. If, for example proteins, enzymes, peptides, etc. are being sold, it is necessary to ensure that the products sold are free from proteases, so that the product sold does not break down on its own. The same can be used for testing the specific activity of proteases, e.g. after prolonged storage, regarding the question as to whether the enzymes are still active.
The proteolytic enzymes are divided into endopeptidases (proteases) and exopeptidases. Endopeptidases cleave amino linkages inside peptides. Exopeptidases digest peptides amino acid by amino acid starting from their ends, i.e. they cleave terminal amino acids. The exopeptidases can be further subdivided into aminopeptidases and carboxypeptidases, as claimed in their activity at the amino or N end or at the carboxy or C end of the peptide.
For determination of the concentration or activity of various peptidases and proteases, to date various fluorescence-based tests have been developed, and their operating principles and sensitivities are briefly described below.
One possibility for detecting proteolytic enzymes is offered by the “ENZCHEK® Protease Assay Kit” from the company Molecular Probes (Eugene, USA). For detecting proteases, this kit employs casein derivatives, to which very many pH-insensitive green or red fluorescing BODIPY® dyes are coupled (Karolin J., Johansson B. A., Strandberg L., Ny T.; J. Am. Chem. Soc., 1994, 116, 7801-7806). On account of the very high degree of marking of the protein, the intermolecular distances between the fluorophores are very small (typically of the order of a few nanometers). The dyes therefore extinguish each other, inter ella by dimer formation. Casein is a large enzyme, which contains many binding sites for proteases, because it has many different sequence segments. If the dye-marked casein derivative comes into contact with proteases, e.g. with trypsin, which hydrolyzes peptide bonds of the basic amino acids arginine and lysine on the carboxy side, these split the casein derivative into smaller peptides, so that as a rule the distance between the dyes increases. Extinction does not occur, and an increase in fluorescence is observed, indicating that proteolytic enzymes are present. For trypsin, for example, the detection sensitivity of this kit is a few ng/ml. For these small quantities of protease the detection time or measurement time is more than 10 hours. This test detects many proteolytic enzymes nonspecifically, since casein is digested by elastase, pepsin, thermolysin, papain, trypsin, etc.
An enzyme test based on Rhodamine 110 for various proteases and peptidases (Leytus S. P., Melhado L. L., Mangel W. F.: Rhodamine-based compounds as fluorogenic substrates for serine protease, (1983) Biochem. J., 209 (2): 299-307; Leytus S. P., Patterson W. L., Mangel W. F.: New class of sensitive and selective fluorogenic substrates for serine proteinases. Amino acid and dipeptide derivatives of rhodamine, (1983) Biochem. J., 215 (2): 253-260) is also being marketed for the detection of serine proteases. In this test, at least one of the amino groups of Rhodamine 110 is coupled covalently to an amino acid (usually arginine), causing strong extinction of the fluorescence of the fluorophore. After digestion of the peptide bonds there is a dramatic increase in fluorescence of Rhodamine 110. For caspase-3, the sensitivity of the test is a few ng absolute quantity of enzyme. Disadvantages are that the principle is not of general applicability, sometimes its action is nonspecific, and it is relatively insensitive.
A protease or peptidase test can also be accomplished by means of Forster fluorescence resonance energy transfer (FRET) (Forster Th.: Zwischenmolekulare Energiewanderung und Fluoreszenz [Intermolecular Energy Transfer and Fluorescence], (1948) Annalen der Physik, 2: 55-75). In this, the specific identification sequence of an endopeptidase is marked covalently at both ends with a donor dye and an acceptor dye. Owing to spectral overlap of donor emission with acceptor absorption, the fluorescence of the donor is extinguished at small distances. If the peptide is cleaved by the endopeptidase, the spatially close contact between donor and acceptor is lost, leading to an increase in fluorescence. Detection of HIV protease will be briefly explained here, as an example. The specific cleaving sequence of HIV protease is coupled, near the cleavage site, with an
The peptide sequence is then: Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys-(DABCYL)-Arg SEQ ID NO: 1, using the 3-letter abbreviations for the amino acids. After cleavage by the HIV protease we have two parts: Arg-Glu(EDANS)Ser-Gln-Asn-Tyr-OH SEQ ID NO: 2 and Pro-Ile-Val-Gln-Lys(DABCYL)-Arg SEQ ID NO: 3. Suitable small distance between the two dyes. Sensitivity is of the order of nanomolar solutions of HIV protease. A disadvantage is the expensive synthesis of the substrate with specific coupling of a donor and an acceptor. If the substrate is to some extent marked incompletely, e.g. with just one donor, this leads to poor sensitivity of the test.
The aim of the invention is to improve the possibilities for a specific detection of proteolytic enzymes.
This aim is achieved by the inventions as claimed in the independent claims. Advantageous developments are described in the subclaims.